Procaine salts of biosynthetic penicillins



Patented Oct. 31, 1 950 T v. UNITED STATES PATENT OFFICE PROCAINE SALTSF BIOS YNTHETIQ PENICILLINS Harley W. Rhodehamel, lin, Indianapolis,Ind., assignor to Eli Lilly and Company, Indianapolis, Ind., acorporation of Indiana a No Drawing. Application July 1, 1948, SerialNo. 36,458

4 Claims. (01. zoo-302) 1 2 My invention relates to salts ofbiosynthetic g thetic penicillin may be reacted with a soluble acidpenicillins and more particularly to insoluble addition salt ofprocaine. Thus, for example,

salts of cycloaliphatic methyl penicillins. the acidic form of thebiosynthetic penicillin and An object of my invention is to prepareinprocaine base are mixed together in amyl acesoluble salts ofcycloaliphatic methyl penicillins. 5 tate to yield as a precipitate, thedesired pro- Another object is to provide insoluble salts usecaine saltof the biosynthetic penicillin. Alterful in the isolation andpurification of biosynnatively, the sodium salt of the biosyntheticthetic penicillins. Another object is to provide penicillin and procainehydrochloride are reinsoluble salts of cycloaliphatic methylpenicilacted in aqueous solution to yield the insoluble lins which havea prolonged therapeutic effect. procaine salt. The insoluble salt whichprecipi- Other objects will be apparent from the followtates isisolated, as by filtration, and freed from ingdisclosure of myinvention. traces of solvent in any suitable manner, such I havediscovered that cycloaliphatic methyl as subjecting the salt to avacuum. penicillins form crystalline salts with. procaine It isdesirable to employ an amount of proand that such salts possessproperties whereby caine salt or p oc b in Slight StO Ch O- the aboveand other objects are accomplished. metric excess to assure the completereaction.

The novel salts of my invention may be rep of all of the biosyntheticpenicillin present. The r s nt d by th following formula, procaine saltsof this invention being substanwherein R represents a member of thegroup tially insolublein water and the common organic consisting ofcycloaliphatic hydrocarbon radisolvents, precipitate when prepared inone of cals having from 5 to 6 carbon atoms in the ring, these solvents,leaving the excess procaine or and the lower alkyl, and halogensubstitution soluble salt thereof in. solution. It is believed,

products thereof. that the procaine salt of the biosynthetic penicil-Illustrative examples of compounds within the lin comprises thecombination of the acidic carscope of my invention are the procainesalts of 'boxyl group of the penicillin with the basicnitrocyclopentylmethyl penicillin, 2 cycloh'exenylgen of thediethylaminoethyl group in the promethyl penicillin, 4methylcyclohexylmethyl caine molecule. penicillin, 3-chlorocyclopentylmethyl penicillin The biosynthetic penicillin may berecovered and l-ethyl-2-cyclohexenylmethyl penicillin." from itscombination with procaine by dissolv- The cycloaliphatic methylpenicillins :used in' mg or suspendin h p a n Salt in q s producing thesalts of m invention may be 01;;- solution, acidifying: the solution toabout pH 2; tained by growing a penicillin-producing mold and extractingthe biosynthetic penicillin with a in a, culture medium containing a,suit bl prepenicillin solvent such as amyl acetate, chlorocursorcompound which the mold may metabolize form and the like. .From thesolution thus oband incorporate in the biosynthetic penicillin. 40 d1Jhe biosynthetic penicillin y be r Procedures for the preparation ofthe biosyn-' Covered y y of the eve al Suitable known thetic penicillinsare described in detail in the processespublication by Behrens et al.,J. Biol. Chem. e f l w xampl s d s sp fi p p- 175, 793 (1948). arationsof several salts of my invention.

The above-mentioned procaine salts of cyclo- Em le 1 aliphatic methylpenicillins may be prepared by mp chemical reactions of addition ormetathesis. The procaine salt of cyclopentylmethyl penicil- The acidicform of the biosynthetic penicillin may lin represented by the formulat) 00H be reacted with procaine base, or a soluble metallic is obtainedas follows:

or metalline (e. g. ammonium) salt of the biosyny l p yl yl p in d n ppar 3 4 ing the above salt may be produced by growing acetate containingabout 2.5 g. of procaine base. a penicillin-producing mold in a culturemedium The procaine salt of cyclohexylmethyl penicillin containing about0.02 percent of N-(Z-hydroxyprecipitates in crystalline form. Thesolution is ethyl)-cyclopentylacetamide, and the biosyncooled to aboutC. for about 2 hours and is thetic penicillin produced in the broth maybe 5 then filtered to isolate the precipitate E isolated as the sodiumsalt. salt of cyclohexylmethyl penicillin. The isolated To about 5.2parts of the sodium salt of the crystalline salt is washed with a smallamount of cyclopentylmethyl penicillin dissolved in about cold amylacetate and dried in vacuo to remove 30 parts of water is added asolution of about 5 traces of moisture and residual amyl acetate. parts(20 percent excess) of procaine hydrochlo- The cycl y Penicillin isrecovered ride in parts of water. An immediate crystalfrom its insolubleprocaine salt as follows: 2 g. line precipitate of the procaine salt ofcycloof the procaine salt are suspended in about 50 ml. pentylmethylpenicillin separates. The mixture of cold water and sufilcient dilutesulfuric acid is cooled to about 0 C. for about 2 hours and is is addedslowly to d y the o u o to about then filtered to separate the insolubleprocaine 15 pH 2- The acidified mixture is extracted with salt ofcyclopentylmethyl penicillin. The sepaabout 50 ml. of amyl acetatewhereupon the Cyclorated salt is washed with a small amount of cold h ypenicillin dissolves in the ll water and is then dried in va uo. tate,and the procaine in the form of its sulfate Example 2 salt dissolves inthe aqueous phase. The amyl g0 acetate layer is separated from theaqueous layer The procaine salt of 2-cyclopentenylmethyl and thecyclohexylmethyl penicillin is recovered penicillin represented by thefollowing formula in purified form from the amyl acetate by ex- OOH maybe prepared as follows: traction of the amyl acetate with cold dilute2-cyclopentenylmethyl penicillin is obtained by m aqueous alkali.growing a penicillin-producing mold in a culture By proceeding throughthe insoluble procaine medium containing about 0.02 percent of N-(2--penicillin salt, substantial purification of thecyhydroxyethyl)-2-cyclopentenylacetamide, and is clohexylmethylpenicillin is efiected, the degree isolated as its sodium salt. Theprocaine salt of of purification amounting to percent or more.2-cyclopentenylmethyl penicillin is obtained Ire. more greatly purifiedcyclohexylmethyl penifrom equal proportions of the sodium salt of 2-cillin is desired, the procaine salt of the penicilcyclopentenylmethylpenicillin and procaine hylin is recrystallized from alcohol or othersuitdrochloride by the procedure described in Exable solvent prior tothe recovery of the biosynample 1. thetic penicillin therefrom. Recoveryof the cy- Example 3 W clohexylmethyl penicillin from the purifiedprocaine salt ields the bios thetic enicillin in Instead of using asoluble salt of the penicillin substantian; pure form yn p and aprocaine salt as described in Examples 1 and 2, the reaction of the acidform of the bio- Example 4 Synthetic penicillin and the procaine basemay be 4 5 The procaine salt of 3-chlorocyclopentylmethyl' used. Thebiosynthetic penicillin in acid form is penicillin hi h may brepresented b t folobtained by extraction of the acidified broth. lo informula lL-1LLH-H2N-G O 0 (CH2) 2N C2115) A 0 0 on The procaine salt ofcyclohexylmethyl penicilis obtained as'follows: lin represented by thefollowing formula A penicillin-producing mold is grown in a cul- Ii 0OOH may be prepared as follows: cyclohexylmethyl ture medium containingabout 0.02 percent of the penicillin is obtained by growing apenicillin-prosodium salt of 3-chlorocyclopentylacetic acid, and ducingmold in a culture medium containing the 3-ch10r0cyc10penty1methyl p ni pabout 0.02 percent of N-(2-hydrcxyethyl)-cyc1oduced in the broth isisolated as its potassium hexylacetamicle, and extracting the culturemedium or broth with amyl acetate The amyl To about one part of thepotassium salt of the tate is clarified with about 0.5 percent ofdecolor- 3'chlomcyclopentylmethyl penicillin dissolved in izing carbonor fullers earth and is concentrated about 6 garts of i 1s aqded ofabout by evaporation in vacuo. To about 4 liters of fi q f i m 3 parts.of this amyl acetate solution containin about 3 wa er. 6 crys a1 me p1eclpltat-e 9f the-procame g salt of 3-cyclopentylmethyl penicillin whichsepa- Of cycloheXylmethyl iii-K110111111 afiidic form are rates isremoved by filtration, washed with a small added slowly and withstirring v100 cc. of amyl amount of cold water and dried in vacuo.

2;-528,-177 N 5- 611 Example 5 will be readily understood, theparticlesize of the salt in suspension should be suificiently small to Theprocaine salt of 4-ethylcyclohexylmethyl permit the particles to passeasily through the penicillin represented by the following formula lumenof a hypodermic needle without packing OOH may be prepared from thesodium salt of 4-ethylwithin and blocking the needle. Illustratively,

cyclohexylmethyl penicillin and procaine hydroa particle size whichavoids blocking ofaIZO- chloride according to the procedure described ingauge needle is that which will pass through a Example 4. I ZOO-meshscreen.

Th -e h y y1m y p lli used n l By wa or illustration, an aqueousmedicinal the p paration is obtained by growi a p composition which uponintramuscular injection lin-produeing m d i the presence of about iscapable of maintaining a therapeutically efpercent f y y y y fectivebiosynthetic penicillin blood level for a. hexylacetamldeperiod of about24 hours comprises a suspension Example 6 containing about 300 mg. offinely divided pro- The procaine p; of 2..methy1cyc1openty1- caine saltof cyclopentylmethyl penicillin per ml. methyl penicillin represented bythe following of Waterformma, qsince, as is known, penicillins arerelatively OOH is prepared from the sodium salt of Z-methylcyunstable inthe presence of appreciable amounts clopentylmethyl penicillin andprocaine hydroof moisture, aqueous compositions containing the chloridein water solution substantially accordnovel procaine salts of thisinvention should be ing to the procedure described in Example 1. usedwithin a comparatively short time after The sodium salt of the2-methy1cyclopenty1- their preparation. For this reason it is oftendemethyl penicillin used may be obtained by growsirable to provide theprocaine salts in oleagiing a penicillin-producing mold in the presencenous vehicles. A suitable oleaginous composition of a small amount of awater-soluble salt of cyclowhich will retain therapeu c p y Over a longpentylacetic acid. period of time is prepared by suspending in aboutExample 7 5 ml. of sesame oil 2 g. of the procaine salt ofcyclopentylmethyl penicillin, ground to a suffl- The procaine salt of2-cyclohexenylmethyl ciently small particle size so that all of themapenicillin represented by the following formula terial will passthrough a ZOO-mesh screen. In-

OOH

may be prepared from the potassium salt of 2- tramuscular injection of 1ml. of this suspension cyclohexenylmethyl penicillin and procainesulwill maintain a therapeutically effective blood fate'in watersolution substantially according to concentration of cyclopentylmethylpenicillin for the procedure described in Example 1. at least 24 hours.

The potassium salt of the 2-cyclohexenylmethyl It is to be understoodthat the above-menpenicillin used may be obtained by growing apenitioned therapeutic compositions are given by way cillin-producingmold in a culture medium conof illustration only and it will be apparentto taining about 0.02 percent of N-(Z-hydroxythose skilled in the artthat numerous modificaethyl) -2-cyclohexenylacetamide, and isolatingtions and variations of the above medicinal comthe penicillin as thepotassium salt. positions may be made.

For therapeutic purposes, the procaine salts of I claim as my invention:the biosynthetic penicillins of this invention pref- 1. The procainesalt of a cycloaliphatic methyl erably are administered intramuscularlyas finely penicillin represented by the formula coon divided suspensionsof the salts in liquid vehicles. in which R represents a, member of thegroup Suitable vehicles for the provision of injectable consisting ofcycloaliphatic hydrocarbon radisuspensions include aqueous andoleaginous vecals having from 5 to 6 carbon atoms in the ring,

hicles, for example, water, and vegetable oils such and thelower alkyl,and halogen substitution as cottonseed oil, sesame oil, and the like. Asproducts thereof.

21 The-procaine salt" of cyclopentylmethyl penicillin represented by theformula 3. The procaine salt of cyclohexylmethyl penicillin representedby the formula 4. The procaine salt of 2-cyc1ohexenylmethyl penicillinrepresented by the formula,

0 COOH HARLEY W. RHODEHANIEL, JR;

REFERENCES CITED

1. THE PROCAINE SALT OF A CYCLOALIPHATIC METHYL PENICILLIN REPRESENTEDBY THE FORMULA